Compositions and methods for promoting patency of vascular grafts

ABSTRACT

Methods for increasing the patency of biodegradable, synthetic vascular grafts are provided. The methods include administering one or more cytokines and/or chemokines that promote outward tissue remodeling of the vascular grafts and vascular neotissue formation. The disclosed methods do not require cell seeding of the vascular grafts, thus avoiding many problems associated with cell seeding. Biodegradable, polymeric vascular grafts which provide controlled release of cytokines and/or chemokines at the site of vascular graft implantation are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and benefit of U.S. ProvisionalPatent Application No. 61/010,406 filed on Jan. 8, 2008, by ChristopherK. Breuer, Themis R. Kyriakides and Jason D. Roh, and where permissibleis incorporated by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant number5K08HL083980 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates generally to compositions and methods forincreasing the patency of vascular grafts, in particular vascular graftsmade from biodegradable, polymeric scaffolds.

BACKGROUND OF THE INVENTION

Cardiac defects are the most common congenital anomalies affectingnearly 1% of all live births. Despite major advances in the treatment ofcongenital heart disease (CHD), it remains the leading cause of deathdue to congenital anomalies in the newborn period. CHD results from amyriad of structural anomalies that present over a broad spectrum.Single ventricle anomalies make up one of the largest groups of cardiacanomalies resulting in severe, life-threatening disease. Singleventricle anomalies are made up of a group of cardiac defects that aredramatically different from each other structurally, but share a commonfeature that only one of two ventricles is of adequate functional size.Some of the cardiac anomalies that result in single ventricle physiologyinclude: tricuspid atresia, pulmonary atresia, and hypoplastic leftheart syndrome. This group of congenital cardiovascular anomaliesresults in mixing of deoxygenated blood from the pulmonary circulationwith oxygenated blood from the systemic circulation. The circulation ofdeoxygenated blood through the systemic circulation causes chronichypoxia and cyanosis, a bluish discoloration of the skin resulting inthe moniker “blue baby syndrome”. Mixing of blood between the pulmonaryand systemic circulation can also cause volume overload to the ventriclethat if untreated can lead to heart failure. Untreated single ventriclecardiac anomalies are associated with 70% mortality during the firstyear of life (Samanek, Pediatr. Cardiol., 13:152-8 (1992)). Thetreatment of choice for single ventricle anomaly is surgicalreconstruction (Giannico, et al., J. Amer. College Card., 47(10):2065-73(2006); Petrossian, et al., J. Thorac. Cardiovasc. Surg., 132:1054-63(2006)). Without surgery, survival into adulthood is unusual (Hager, etal., J. Thorac. Cardiovasc. Surg., 123:1214-7 (2002)).

Despite the dramatic structural differences in the cardiac defectscausing single ventricle physiology, the ultimate plans for stagedsurgical reconstruction are actually quite similar. The goal of thisseries of staged operations is to separate the pulmonary circulationfrom the systemic circulation. This eliminates the mixing of systemicand pulmonary blood flow, resulting in improved systemic oxygenation,and preventing volume overload, thus normalizing the volume work of thesystemic ventricle, thereby preventing heart failure. This isaccomplished through a series of staged operations designed toreconstruct the cardiovascular structures so that the single ventriclepumps oxygenated blood through the systemic circulation. Thedeoxygenated blood is then passively circulated through the pulmonarycirculation where it is oxygenated and returned to the heart. This typeof surgical procedure is referred to as a Fontan operation. The Fontanoperation has undergone several modifications since it was firstreported in 1971 (Fontan, et al., Thorax, 26(3):240-8 (1971)). The mostcommonly performed modification of the Fontan operation is the extracardiac total cavopulmonary connection (EC TCPC). The modified Fontanoperation is considered the standard of care for the treatment ofpatients with single ventricle cardiac anomalies and has substantiallyimproved both the quality and long-term survival of these patients.However; it is still considered a palliative (non-curative) procedurewith significant morbidity and mortality (Giannico, et al., J. Amer.College Card., 47(10):2065-73 (2006); Petrossian, et al., J. Thorac.Cardiovasc. Surg., 132:1054-63 (2006)). One important cause of morbidityand mortality in patients requiring the Fontan operation is the conduitused to connect the inferior vena cava to the right pulmonary arterywhen native tissue cannot be used (Jonas, et al., J. Thorac. Cardiovasc.Surg., 117:688-96 (1999)). When Fontan and Kirklin reviewed the lateoutcome of an early cohort of patients surviving the Fontan procedure,they concluded that much of the late morbidity could be attributed toproblems associated with conduit use (Fontan, et al., Circulation,81:1520-36 (1990)). It is widely accepted that the ideal conduit has notyet been developed (Conte, FASEB, 12:43-5 (1998); Kakisis, J. VasaSurg., 41:349-54 (2005)). Polytetrafluoro-ethylene (PTFE or Gore-Tex®)conduits are currently the most widely used vascular grafts for EC TCPC(Petrossian, et al., J. Thorac. Cardiovasc. Surg., 132:1054-63 (2006)).Use of other synthetic conduits or even biological vascular grafts isdescribed in the literature but to a much more limited extent comparedto PTFE (Petrossian, et al., J. Thorac. Cardiovasc. Surg., 117:688-96(1999)).

While data describing the long-term graft failure rates for conduitsused for EC TCPC is limited, long-term data regarding use of both valvedand unvalved conduits for other similar congenital heart operations arewidely available and are poor (Dearani, et al., Ann. Thorac. Surg.,75:399-411 (2003)). Late problems include conduit degeneration withprogressive obstruction, lack of growth potential, increasedsusceptibility to infection and increased risk for thrombo-emboliccomplications. Both synthetic and biological conduits are used for theseoperations. PTFE and other synthetic conduits such as Dacron lack growthpotential, necessitating re-operation when a patient outgrows thevascular graft. Synthetic conduits are a significant cause ofthrombo-embolic complication due to the large area of synthetic materialin contact with blood, which causes activation of the coagulationcascade (Petrossian, et al., J. Thorac. Cardiovasc. Surg., 117:688-96(1999)). Other clinically available conduits including biological graftssuch as homografts and heterografts are associated with significantlylower thromboembolic complication rates compared to synthetic grafts,however; they too lack growth potential and unfortunately have poordurability due to their propensity for accelerated calcific degradationand secondary graft failure (Stark, Peadiatr. Cardiol., 19:282-8 (1998);Cleveland, et al., Circulation, 86(suppl II):II150-3 (1992); Jonas, etal., Circulation, 72(suppl II):II77-83 (1985)). These grafts tend tobecome stenotic and calcify. This process seems to be immune mediatedand more aggressive in younger patients (Karamlou, et al., Eur. J.Cardiothorac. Surg., 27:548-53 (2005)). It is basically assumed that allsuch conduits will eventually need to be replaced (Bermudez, et al.,Ann. Thorac. Surg., 77:881-8 (2004)). Re-operations are associated withsignificant morbidity and mortality with early post-operative mortalityrates around 5% in the best centers (Dearani, et al., Ann. Thorac.Surg., 75:399-411 (2003)). Early and midterm results for these graftsare variable with 5 year patency rates between 65-90%. Long-term datademonstrating graft failure rates between 70-100% at 10-15 years havebeen reported (Jonas, et al., Circulation, 72(suppl II):II77-83 (1985);Peadiatr. Cardiol., 19:282-8 (1998); Homann, et al., Eur. J.Cardiothorac. Surg., 17:624-30 (2000)). Primary determinants of graftfailure include size (with an increased rate of failure in grafts lessthan 18 mm with another significant drop off below 15 mm) andre-operation (with primary grafts performing better than replacementgrafts) (Homann, et al., Eur. J. Cardiothorac. Surg., 17:624-30 (2000)).The best long-term results have been obtained when autologous tissue hasbeen used for or incorporated into the conduit with long-term patencyrates exceeding 80% (Bermudez, et al., Ann. Thorac. Surg., 77:881-8(2004)).

Autografts, conduits created from an individual's own (autologous)tissue, have better long-term effectiveness than any synthetic orbiological conduit currently available for use in pediatriccardiovascular surgical applications (Dearani, et al., Ann. Thorac.Surg., 75:399-411 (2003); Bermudez, et al., Ann. Thorac. Surg., 77:881-8(2004)). Unfortunately autografts are limited in supply, necessitatingthe use of synthetic or biological conduits in most cases (Homann, etal., Eur. J. Cardiothorac. Surg., 17:624-30 (2000)). Use of synthetic orbiological vascular grafts result in increased graft failure rates andincreased morbidity and mortality rates when compared to similaroperations performed using autologous tissue (Jonas, et al.,Circulation, 72(suppl II):II77-83 (1985); Bermudez, et al., Ann. Thorac.Surg., 77:881-8 (2004)).

Complications arising from the use of currently available vasculargrafts are a leading cause of postoperative morbidity and mortalityafter congenital heart surgery (Jonas, et al., J. Thorac. Cardiovasc.Surg., 117:688-96 (1999)). Additionally the lack of growth potential ofall currently available vascular conduits is problematic(Alexi-Meskishvili, et al., Eur. J. Cardiothorac. Surg., 18:690-5(2000)). Use of over-sized grafts in an attempt to avoid outgrowing aconduit is widely practiced. Postponing surgery until the patient isbetween 2 and 4 years of age, when the diameter of the IVC approaches60-80% of the adult frequently enables placement of near adult sizedconduits (20-22 mm) and limits the need for conduit replacement based onsomatic growth alone, however; graft over-sizing is associated with anincreased risk of complications (Alexi-Meskishvili, et al., Eur. J.Cardiothorac. Surg., 18:690-5 (2000)). Delaying surgery to minimize thenumber of re-operations can lead to cardiac dysfunction or even heartfailure due to prolonged exposure to volume overload and chronic hypoxia(Petrossian, et al., J. Thorac. Cardiovasc. Surg., 117:688-96 (1999)).Additionally, recent studies have demonstrated marked improvement insomatic growth in patients who undergo surgery at an earlier age,providing further support for the performance of EC TCPC at an earlierage (Ovroutski, et al., Eur. J. Cardiothorac. Surg., 26:1073-9 (2004)).The upper limit of over-sizing is approximately 1.5 times the size ofthe native vessel after which point over-sizing will cause substantialnegative hemodynamic consequences (Lardo, et al., J. Thorac. Cardiovasc.Surg., 117:697-704 (1999)). Recent studies recommend limitingover-sizing conduits to 1.2 times the size of the native vessel becauseit is thought that the increased risk of thrombo-embolic complicationsassociated with the use of over-sized grafts is greater than the risk ofconduit replacement (Alexi-Meskishvili, et al., Eur. J. Cardiothorac.Surg., 18:690-5 (2000)). The development of a vascular graft with growthpotential would eliminate this problem and have dramatic implicationsfor the field of congenital heart surgery.

Tissue engineering offers a strategy for constructing autologous graftsand thereby increasing the pool of potential autografts. Using theclassical tissue engineering paradigm, autologous cells can be seededonto a biodegradable tubular scaffold. The scaffold provides sites forcell attachment and space for neotissue formation (Langer and Vacanti,Science, 260:920-6 (1993)). The resulting neotissue can be used forreconstructive surgical applications such as creation of a vasculargraft for use in pediatric cardiothoracic operations (Shinoka, et al.,J. Thorac. Cardiovasc. Surg., 115:536-46 (1998)). Extensive large animalstudies using both ovine and canine animal models, have demonstrated thefeasibility of using tissue engineering methodology to constructconduits for use as large caliber grafts in the venous or pulmonarycirculation (Shinoka, et al., J. Thorac. Cardiovasc. Surg., 115:536-46(1998); Watanabe, et al., Tissue Eng., 7(4):429-39 (2001); Matsumura, etal., Biomaterials, 24:2303-8 (2003); Matsumura, et al., Tissue Eng.,12:1-9 (2006)).

Many studies using biodegradable, synthetic scaffolds have employedvascular cells that were isolated from autologous vessel biopsies. Morerecent studies have explored the use of autologous cells obtained frombone marrow aspirate Matsumura, et al., Biomaterials, 24:2303-8 (2003)).Based in part on the success of animal studies and on the great promiseassociated with the development of a vascular graft with growthpotential for congenital heart surgery, a pilot clinical study wasconducted to evaluate the feasibility and safety of using tissueengineered vascular grafts as conduits for EC TCPC in patients withsingle ventricle cardiac anomalies (Shinoka, et al., New Engl. J. Med.,344(7):532-3 (2001)); Naito, et al., J. Thorac. Cardiovasc. Surg.,125:419-20 (2003)). To date 25 TEVG have been implanted as conduits forEC TCPC with follow-up out through seven years (Shinoka, et al., J.Thorac. Cardiovasc. Surg., 129:1300-8 (2005)). The tissue engineeredvascular grafts functioned well without evidence of graft failure. Nograft has had to be replaced. There has been no graft related mortality.There have been two graft related complications, which include thedevelopment of significant stenosis in two small diameter (<18 mm)conduits. Both were successfully treated, the first with angioplasty andthe second with angioplasty and stenting. There were no reportedthromboembolic or hemorrhagic complications, infectious complication orevidence of aneurysm formation. Additionally serial imaging demonstratedthe growth potential of these grafts. These data support the overallfeasibility and safety of this technology.

The methodology of seeding synthetic vascular grafts with autologouscells, however, is still problematic for many reasons. First, itrequires an invasive procedure (biopsy) in addition to the need for asubstantial period of time in order to expand the cells in culture thatlimited its clinical utility. This approach also faces the inherentdifficulty in obtaining healthy autologous cells from diseased donors(Poh, et al., Lancet, 365:2122-24 (2005); Solan, et al., CellTransplant., 14(7):481-8 (2005)). The use of cell culture also resultsin an increased risk of contamination and even the potential fordedifferentiation of the cultured cells. The use of autologous cells toseed the polymeric grafts also limits the off-the-shelf availability oftissue engineered vascular grafts, thereby limiting their overallclinical utility.

Therefore, it is an object of the invention to provide methods forincreasing the patency of biodegradable, synthetic vascular graftswithout using cell seeding.

It is another object of the invention to provide biodegradable,synthetic vascular grafts with growth potential that have increasedpatency.

SUMMARY OF THE INVENTION

Methods for increasing the patency of biodegradable, synthetic vasculargrafts are provided. The methods include administering one or morecytokines and/or chemokines that promote outward tissue remodeling ofthe vascular grafts and vascular neotissue formation. The polymericvascular grafts are tubular, porous structures that allow forrecruitment and integration of host cells into the graft that mediateremodeling and vascular neotissue formation. The vascular grafts arebiodegradable, which allows for the grafts to be completely replaced byforming neotissue as they degrade. The methods do not require cellseeding of the vascular grafts, avoiding many problems associated withseeding, including the need for an invasive procedure to obtainautologous cells, the need for a substantial period of time in order toexpand the cells in culture, the inherent difficulty in obtaininghealthy autologous cells from diseased donors, and an increased risk ofcontamination and the potential for dedifferentiation of the cells. Thedisclosed biodegradable, synthetic vascular grafts therefore have agreater off-the-shelf availability and increased overall clinicalutility.

The biodegradable, polymeric vascular grafts may be fabricated frombiodegradable polymers using any known method. In one embodiment, thepolymeric vascular grafts are fabricated from woven or non-woven sheetsor felts or polymeric fibers. The polymers and fabrication methods areselected to produce vascular grafts with biomechanical properties, suchas initial burst pressure, suture retention strength, elasticity andtensile strength, suitable for use as vascular conduits. Polymeric wovenor non-woven sheets or felts may be further treated with polymericsealants to modify the biomechanical properties of the graft and/or tocontrol the total porosity and pore size distribution range of thevascular graft.

It is believed that cytokines and chemokines function to increase thepatency of polymeric vascular grafts by causing the recruitment of hostcells to the graft that promote vascular remodeling and vascularneotissue formation. Suitable cytokine and/or chemokines include thosethat promote the recruitment of host cells to the implanted polymericvascular grafts. Particularly suitable cytokines and chemokines includethose that promote early recruitment of monocytes to the implantedpolymeric vascular grafts. Exemplary cytokines or chemokines include,but are not limited to, monocyte chemoattractant protein-1 (MCP-1),interleukin-1beta (IL-1β), and granulocyte colony-stimulating factor(G-CSF or GCSF). Additional bioactive agents that promote adaptation ofthe vascular graft may also be administered.

Cytokines and/or chemokines may be administered in an effective amountto prevent, inhibit or reduce restenosis, thrombus or aneurysm formationin implanted polymeric vascular grafts. Cytokines and/or chemokines maybe administered prior to vascular graft implantation, at the same timeas vascular graft implantation, following vascular graft implantation,or any combination thereof. In one embodiment, cytokines or chemokinesare administered either locally or systemically from a controlledrelease formulation.

In a preferred embodiment, cytokines or chemokines are administeredlocally at the site of graft implantation using a controlled releaseformulation. In one embodiment, the cytokine or chemokine isincorporated into or onto the polymeric vascular graft which functionsas a controlled release formulation. The cytokine or chemokine may bedispersed evenly throughout the polymeric vascular graft using any knownsuitable method. In another embodiment, the cytokine or chemokine may beencapsulated in a second polymeric matrix that is incorporated into thepolymeric vascular graft. In one embodiment, the cytokines or chemokinesare encapsulated into microspheres, nanospheres, microparticles and/ormicrocapsules, and seeded into the porous vascular graft.

The biodegradable, synthetic vascular grafts may be used as venous,arterial or artero-venous conduits for any vascular or cardiovascularsurgical application. Exemplary applications include, but are notlimited to, congenital heart surgery, coronary artery bypass surgery,peripheral vascular surgery and angioaccess.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the difference in patency rates (percentpatent) between seeded and unseeded tissue engineered vascular grafts(TEVGs) at 24 weeks after implantation.

FIG. 2 is a line graph showing the fraction of human RNA (GAPDH)remaining in the TEVG as a function of days following graftimplantation. RNA quantitation was performed using Q-RT-PCR. The hatchedarea represents the Q-RT-PCR limit of detection.

FIG. 3 is a bar graph showing the number of F4/80 positive mousemonocytes/macrophages per cell-seeded (human bone marrow cell (hBMC)) orunseeded scaffold at week 1 following scaffold implantation.

FIG. 4A is a bar graph showing the concentration (pg/ml) of IL-1 betaproduced by hBMCs when seeded onto scaffolds or unseeded, as determinedby ELISA. FIG. 4B is a bar graph showing the concentration (pg/ml) ofMCP-1 produced by hBMCs when seeded onto scaffolds or unseeded, asdetermined by ELISA.

FIG. 5A is a bar graph showing the internal diameter (mm) of TEVGs at 10weeks after implantation. TEVGs were unseeded, seeded with BMCs fromwild-type mice, or seeded with cells from MCP-1 knockout mice. FIG. 5Bis a bar graph showing the wall thickness (mm) of TEVGs at 10 weeksafter implantation. TEVGs were unseeded, seeded with BMCs from wild-typemice, or seeded with cells from MCP-1 knockout mice.

FIG. 6 is a line graph showing the release profile of MCP-1 (ng) fromMCP-1 eluting scaffolds as a function of time (hours).

FIG. 7A is a bar graph showing the number of F4/80 positive mousemonocytes/macrophages per scaffold at week 1 following scaffoldimplantation. FIG. 7B is a bar graph showing the internal diameter (mm)of scaffolds at 10 weeks after implantation. FIG. 7C is a bar graphshowing the wall thickness (mm) of scaffolds at 10 weeks afterimplantation. Scaffolds were unseeded, seeded with hBMCs, or unseededwith MCP-1 eluting microspheres.

FIG. 8A is a bar graph showing the internal diameter (mm) of scaffoldsat 10 weeks after implantation for mice treated with G-CSF or untreated.FIG. 8B is a bar graph showing the wall thickness (mm) of scaffolds at10 weeks after implantation for mice treated with G-CSF or untreated.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The terms “conduit”, “graft”, “vascular graft” and “scaffold” are usedinterchangeably herein.

“Restenosis”, as defined herein, means a narrowing of the lumen of ablood vessel at a previously stenotic site (i.e., the site of ballooninflation during angioplasty), or narrowing of the lumen of a bloodvessel or synthetic graft following an interventional procedure (e.g.,narrowing of the venous side of an arterial-venous anastomosis followingbypass surgery using a graft). Restenosis, as used herein, encompassesocclusion. Restenosis includes any luminal narrowing that occursfollowing an injury to the vessel wall. Injuries resulting in restenosiscan therefore include trauma to an atherosclerotic lesion (as seen withangioplasty), a resection of a lesion (as seen with endarterectomy), anexternal trauma (e.g., a cross-clamping injury), or a surgicalanastomosis.

“Copolymer” is used herein to refer to a single polymeric material thatis comprised of two or more different monomers. The copolymer can be ofany form, such as random, block, graft, etc. The copolymers can have anyend-group, including capped or acid end groups.

“Biocompatible” as used herein refers to a material and any metabolitesor degradation products thereof that are generally non-toxic to therecipient and do not cause any significant adverse effects to thesubject.

“Biodegradable” refers to a material that will degrade or erode underphysiologic conditions to smaller units or chemical species that arecapable of being metabolized, eliminated, or excreted by the subject.

“Controlled release” or “modified release”, as used herein, refers to arelease profile in which the active agent release characteristics oftime course and/or location are chosen to accomplish therapeutic orconvenience objectives not offered by conventional dosage forms such assolutions, suspensions, or promptly dissolving dosage forms. Delayedrelease, extended release, and pulsatile release and their combinationsare examples of modified release.

“Bioactive agent” or “active agent” is used herein to refer totherapeutic, prophylactic, and/or diagnostic agents. It includes withoutlimitation physiologically or pharmacologically active substances thatact locally or systemically in the body. A biologically active agent isa substance used for, for example, the treatment, prevention, diagnosis,cure, or mitigation of disease or disorder, a substance which affectsthe structure or function of the body, or pro-drugs, which becomebiologically active or more active after they have been placed in apredetermined physiological environment. Bioactive agents includebiologically, physiologically, or pharmacologically active substancesthat act locally or systemically in the human or animal body. Examplescan include, but are not limited to, small-molecule drugs, peptides,proteins, antibodies, sugars, polysaccharides, nucleotides,oligonucleotides, aptamers, siRNA, nucleic acids, and combinationsthereof. “Bioactive agent” includes a single agent or a plurality ofbioactive agents including, for example, combinations of two or morebioactive agents.

The terms “individual”, “host”, “subject”, and “patient” are usedinterchangeably herein.

II. Methods for Promoting Patency of Biodegradable, Synthetic VascularGrafts

Patency of biodegradable, synthetic vascular grafts is increased byadministering one or more cytokines and/or chemokines to promotelong-term patency of biodegradable, synthetic vascular grafts. Theadministration of cytokines or chemokines increases the patency of thebiodegradable, synthetic vascular grafts relative to the patency of thegrafts in the absence of the cytokine or chemokine.

It is believed that cytokines and chemokines promote patency ofbiodegrabable, synthetic vascular grafts by promoting the earlyrecruitment of host cells, including monocytes, to the vascular graftthat promote outward vascular tissue remodeling and vascular neotissueformation.

The disclosed biodegradable, synthetic vascular grafts do not requirecell seeding to maintain patency of the grafts. This is advantageous,because it avoids problems associated with cell seeding, including theneed for an invasive procedure to obtain autologous cells, the need fora substantial period of time in order to expand the cells in culture,the inherent difficulty in obtaining healthy autologous cells fromdiseased donors, and an increased risk of contamination and thepotential for dedifferentiation of the cells. The disclosedbiodegradable, synthetic vascular grafts therefore have a greater theoff-the-shelf availability and increased overall clinical utility.

A. Polymeric Vascular Grafts

The polymeric vascular grafts disclosed herein are tubular porousconduits fabricated using biodegradable polymers. The pores in thepolymeric vascular grafts allow for recruitment and integration of hostcells into the graft. It is believed that recruited host cells mediateoutward vascular tissue remodeling and vascular neotissue formation.Unlike synthetic vascular grafts that are currently in clinical use, thedisclosed polymeric vascular grafts are biodegradable, which allows forthe grafts to become replaced by forming neotissue as they degrade.Thus, the disclosed polymeric vascular grafts offer growth potentialthat is not possible with currently used synthetic vascular grafts.

The disclosed grafts are substantially tubular in shape with a round orsubstantially round cross section. The tubular grafts have a lumenextending throughout the length of the graft. The grafts may be of anyappropriate length and diameter that is suitable for the intendedsurgical use of the graft. Typically, the graft should be slightlylonger than the length of artery or vein that is to be replaced.

The porous polymeric vascular grafts may be fabricated using anyappropriate method, such as melt processing, solvent processing,leaching, foaming, extrusion, injection molding, compression molding,blow molding, spray drying, extrusion coating, and spinning of fiberswith subsequent processing into woven or non-woven constructs. Pores inthe graft may be derived by any suitable method, including saltleaching, sublimation, solvent evaporation, spray drying, foaming,processing of the materials into fibers and subsequent processing intowoven or non-woven devices. Preferably, the pores of the device arebetween 5 and 500 μm, more preferably between 5 and 250 μm, morepreferably between 5 and 100 μm, in diameter.

In one embodiment, the grafts are formed from a felt or sheet likematerial of the polymer that can be formed into a tubular conduit. Forexample the device could be fabricated as a nonwoven, woven or knittedstructure from extruded polymeric fibers. The polymeric sheet may beformed using any textile construction, including, but not limited to,weaves, knits, braids or filament windings. Any suitable method, such aselectrospinning, may be used to fabricate the nonwoven or wovenpolymeric textile.

The polymers and fabrication methods selected to fabricate the polymericvascular grafts are suitable to produce grafts with biomechanicalproperties suitable for use as vascular conduits. Biomechanicalproperties that are important for vascular graft function includeinitial burst pressure, suture retention strength and elasticity. In oneembodiment, the initial burst pressure of the polymeric vascular graftis between about 1,500 mmHg and about 4,500 mmHg, preferably betweenabout 2,000 mmHg and about 4,500 mmHg. In another embodiment, thepolymeric vascular grafts possess suture retention strengths betweenabout 1 N and about 5 N, preferably between about 2 N and about 4 N. Inanother embodiment, the intrinsic elasticity of the vascular grafts isbetween about 10 MPa and about 50 MPa, preferably between about 15 MPaand about 40 MPa. In another embodiment, the initial tensile strength ofthe vascular grafts is between about 1 MPa and about 10 MPa, preferablybetween about 3 MPa and about 6 MPa.

1. Biodegradable Polymers

The biodegradable, synthetic vascular grafts may be fabricated using anyknown biodegradable polymer, co-polymer, or mixture thereof. Manysuitable biodegradable polymers are known in art.

Examples of preferred biodegradable polymers include synthetic polymersthat degrade by hydrolysis such as poly(hydroxy acids), such as polymersand copolymers of lactic acid and glycolic acid, polyanhydrides,poly(ortho)esters, polyesters, polyurethanes, poly(butic acid),poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates), andpoly(lactide-co-caprolactone). The foregoing materials may be usedalone, as physical mixtures (blends), or as co-polymers.

In a preferred embodiment, the biodegradable, synthetic vascular graftsare fabricated from polyglycolic acid or poly-L-lactic acid. Theexamples below demonstrate that these biodegradable polymers can beextruded into fibers and fabricated into nonwoven felts to producevascular grafts with biomechanical properties suitable for use asvascular conduits, such as tensile strength, elastic modulus and sutureretention strength when combined with appropriate polymeric sealants.These vascular grafts also possess good in vivo biocompatibility andfunctionality.

2. Sealants

Synthetic vascular grafts fabricated from nonwoven, woven or knittedsheets or felts of biodegradable polymers may be further treated withpolymeric sealants. The polymeric sealants function to modify thebiomechanical properties of the vascular grafts, such as tensilestrength and elasticity. Polymeric sealants may also be used to controlthe total porosity and pore size distribution range of the vasculargraft.

Polymeric sealants for the disclosed biodegradable synthetic vasculargrafts may be any biodegradable polymer, including, but not limited to,the list of biodegradable polymers listed above. In one embodiment, thepolymeric sealant is a copolymer of poly(ε-caprolactone) andpoly(L-lactide).

Polymeric sealants may be added to tubular synthetic grafts dissolved inan appropriate solvent to allow for the sealant to penetrate thenonwoven, woven or knitted sheet or felt of biodegradable polymersforming the graft. The polymeric sealant may then be quickly transformedfrom liquid to solid phase by lowering the temperature of the graft.Solvents may then be removed by an appropriate technique, such aslyophilization.

B. Cytokines and Chemokines

Suitable cytokines and chemokines include cytokines and chemokines thatpromote the recruitment of host cells to implanted synthetic vasculargrafts. Particularly suitable cytokines and chemokines include thosethat promote early recruitment of monocytes to implanted syntheticvascular grafts following implantation. It is believed that monocytesthat are recruited early to vascular grafts promote outward vasculartissue remodeling and vascular neotissue formation.

Exemplary cytokines and chemokines include, but are not limited to,interleukin (IL) 1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9,IL-10, IL-12(p40), IL-12(p70), IL-13, IL-15, IL-17, IP-10, eotaxin,interferon γ (IFNγ), granulocyte colony-stimulating factor (G-CSF),granulocyte/macrophage colony-stimulating factor (GM-CSF), monocytechemoattractant protein (MCP-1), macrophage inflammatory protein 1α(MIP-1α), RANTES, tumor necrosis factor (TNF)-α, platelet-derived growthfactor (PDGF)-AA, PDGF-AB/BB, TGF-beta and VEGF, or combinationsthereof. In one embodiment, the cytokine or chemokine is G-CSF. Inanother embodiment, the cytokine or chemokine is MCP-1. In anotherembodiment, the cytokine or chemokine is IL-1β.

The examples below demonstrate that human bone marrow cells (hBMCs),which promote patency when seeded onto synthetic vascular scaffolds,significantly increase production and secretion of MCP-1 and IL-1β whenseeded onto polymeric scaffolds. The examples also demonstrate thatpolymeric vascular grafts seeded with bone marrow cells from MCP-1−/−mice have decreased internal lumen diameters and significantly increasedgraft wall thickness when implanted into mice as compared to polymericvascular grafts seeded with bone marrow cells from wild-type mice. Theexamples also demonstrate that administration of G-CSF prior toimplantation of polymeric vascular scaffolds significantly increases thepatency of the vascular scaffold.

C. Additional Bioactive Agents

Additional bioactive agents that promote vascular graft adaptation mayalso be administered. Suitable bioactive agents or drugs include, butare not limited to: anti-thrombogenic agents such as heparin, heparinderivatives, urokinase, and PPack (dextrophenylalanine proline argininechloromethylketone; anti-proliferative agents such as enoxaprin,angiopeptin, or monoclonal antibodies capable of blocking smooth musclecell proliferation, hirudin, and acetylsalicylic acid;antineoplastic/antiproliferative/anti-mitotic agents such as paclitaxel,5-fluorouracil, cisplatin, vinblastine, vincristine, epothilones,endostatin, angiostatin and thymidine kinase inhibitors; anestheticagents such as lidocaine, bupivacaine, and ropivacaine; anti-coagulantssuch as D-Phe-Pro-Arg chloromethyl keton, an RGD peptide-containingcompound, heparin, antithrombin compounds, platelet receptorantagonists, anti-thrombin antibodies, anti-platelet receptorantibodies, aspirin, prostaglandin inhibitors, platelet inhibitors andtick antiplatelet peptides; vascular cell growth promoters such asgrowth factor inhibitors, growth factor receptor antagonists,transcriptional activators, and translational promoters; vascular cellgrowth inhibitors such as growth factor inhibitors, growth factorreceptor antagonists, transcriptional repressors, translationalrepressors, replication inhibitors, inhibitory antibodies, antibodiesdirected against growth factors, bifunctional molecules consisting of agrowth factor and a cytotoxin, bifunctional molecules consisting of anantibody and a cytotoxin; cholesterol-lowering agents; vasodilatingagents; and agents which interfere with endogenous vascoactivemechanisms.

D. Pharmaceutical Compositions and Methods of Administration

Cytokines or chemokines are administered to a subject receiving apolymeric vascular graft in an effective amount to prevent, inhibit orreduce restenosis, thrombus or aneurysm formation in implanted vasculargrafts. The precise dosage will vary according to a variety of factorssuch as the nature of the particular cytokine(s) or chemokine(s) beingadministered, the route of administration, and subject-dependentvariables (e.g., age, etc.).

Cytokines or chemokines may be administered systemically or locally atthe site of vascular graft implantation. Cytokines or chemokines may beadministered prior to vascular graft implantation, at the same time asvascular graft implantation, following vascular graft implantation, orany combination thereof. In one embodiment, cytokines or chemokines areadministered either locally or systemically from a controlled releaseformulation. The cytokines or chemokines may be administered separatelyfrom additional bioactive agents or may be co-administered.

Pharmaceutical compositions containing peptides or polypeptides (i.e.cytokines and chemokines) may be for administration by parenteral(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), transdermal (either passively or using iontophoresis orelectroporation), or transmucosal (nasal, vaginal, rectal, orsublingual) routes of administration. The compositions can be formulatedin dosage forms appropriate for each route of administration.Compositions containing bioactive agents that are not peptides orpolypeptides can additionally be formulated for enteral administration.

In one embodiment, the cytokine or chemokine is incorporated into oronto the vascular graft. The cytokine or chemokine may be dispersedevenly throughout the polymeric vascular graft using any known suitablemethod. The cytokine or chemokine may be incorporated directly into thevascular graft or may be encapsulated in the form of microspheres,nanospheres, microparticles and/or microcapsules, and seeded into theporous vascular graft.

1. Formulations for Parenteral Administration

In one embodiment, polypeptide bioactive agents, including cytokines andchemokines, are administered in an aqueous solution by parenteralinjection. The formulation may also be in the form of a suspension oremulsion. In general, pharmaceutical compositions are provided includingeffective amounts of a cytokine or chemokine, and optionally includepharmaceutically acceptable diluents, preservatives, solubilizers,emulsifiers, adjuvants and/or carriers. Such compositions includediluents sterile water, buffered saline of various buffer content (e.g.,Tris-HCl, acetate, phosphate), pH and ionic strength; and optionally,additives such as detergents and solubilizing agents (e.g., TWEEN® 20,TWEEN® 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid, sodiummetabisulfite), and preservatives (e.g., Thimersol, benzyl alcohol) andbulking substances (e.g., lactose, mannitol). Examples of non-aqueoussolvents or vehicles are propylene glycol, polyethylene glycol,vegetable oils, such as olive oil and corn oil, gelatin, and injectableorganic esters such as ethyl oleate. The formulations may be lyophilizedand redissolved/resuspended immediately before use. The formulation maybe sterilized by, for example, filtration through a bacteria retainingfilter, by incorporating sterilizing agents into the compositions, byirradiating the compositions, or by heating the compositions.

2. Formulations for Topical Administration

In another embodiment, bioactive agents, including cytokines orchemokines, are administered topically.

Compositions can be delivered to the lungs while inhaling and traverseacross the lung epithelial lining to the blood stream when deliveredeither as an aerosol or spray dried particles having an aerodynamicdiameter of less than about 5 microns.

A wide range of mechanical devices designed for pulmonary delivery oftherapeutic products can be used, including but not limited tonebulizers, metered dose inhalers, and powder inhalers, all of which arefamiliar to those skilled in the art. Some specific examples ofcommercially available devices are the Ultravent nebulizer (MallinckrodtInc., St. Louis, Mo.); the Acorn II nebulizer (Marquest MedicalProducts, Englewood, Colo.); the Ventolin metered dose inhaler (GlaxoInc., Research Triangle Park, N.C.); and the Spinhaler powder inhaler(Fisons Corp., Bedford, Mass.). Nektar, Alkermes and Mannkind all haveinhalable insulin powder inhalers.

Formulations for administration to the mucosa will typically be spraydried drug particles, which may be incorporated into a tablet, gel,capsule, suspension or emulsion. Standard pharmaceutical excipients areavailable from any formulator. Oral formulations may be in the form ofchewing gum, gel strips, tablets or lozenges.

Transdermal formulations may also be prepared. These will typically beointments, lotions, sprays, or patches, all of which can be preparedusing standard technology. Transdermal formulations will require theinclusion of penetration enhancers.

3. Controlled Delivery Polymeric Matrices

In one embodiment, cytokines or chemokines may be administeredsystemically or locally using controlled release formulations. In apreferred embodiment, cytokines or chemokines are administered locallyat the site of graft implantation using a controlled releaseformulation. In one embodiment, the cytokine or chemokine isincorporated into or onto the polymeric vascular graft which functionsas a controlled release formulation. The cytokine or chemokine may bedispersed evenly throughout the polymeric vascular graft using any knownsuitable method. For example, one or more cytokines or chemokines may beadded to the polymeric scaffold during fabrication by adding them to thepolymer solution or emulsion or during the fabrication of a polymerictextile, such as by an electrospinning process. Additionally, oralternatively, one or more cytokines or chemokines may be added to thepolymeric graft following fabrication. In another embodiment, thecytokine or chemokine is preferentially localized to either the exterioror the lumen of the tubular polymeric vascular graft.

In another embodiment, the cytokine or chemokine may be encapsulated ina second polymeric matrix that is incorporated into the polymericvascular graft. In one embodiment, the cytokines or chemokines areencapsulated into microspheres, nanospheres, microparticles and/ormicrocapsules, and seeded into the porous vascular graft. The matrix canbe in the form of microparticles such as microspheres, wherepolypeptides are dispersed within a solid polymeric matrix ormicrocapsules, where the core is of a different material than thepolymeric shell, and the polypeptide is dispersed or suspended in thecore, which may be liquid or solid in nature. Unless specificallydefined herein, microparticles, microspheres, and microcapsules are usedinterchangeably. Alternatively, the polymer may be cast as a thin slabor film, ranging from nanometers to four centimeters, a powder producedby grinding or other standard techniques, or even a gel such as ahydrogel, and used as a coating for the vascular graft.

Either non-biodegradable or biodegradable matrices can be used fordelivery of cytokines or chemokines, although biodegradable matrices arepreferred. These may be natural or synthetic polymers, althoughsynthetic polymers are preferred due to the better characterization ofdegradation and release profiles. The polymer is selected based on theperiod over which release is desired. In some cases linear release maybe most useful, although in others a pulse release or “bulk release” mayprovide more effective results. The polymer may be in the form of ahydrogel (typically in absorbing up to about 90% by weight of water),and can optionally be crosslinked with multivalent ions or polymers.

The matrices can be formed by solvent evaporation, spray drying, solventextraction and other methods known to those skilled in the art.Bioerodible microspheres can be prepared using any of the methodsdeveloped for making microspheres for drug delivery, for example, asdescribed by Mathiowitz and Langer, J. Controlled Release, 5:13-22(1987); Mathiowitz, et al., Reactive Polymers, 6:275-283 (1987); andMathiowitz, et al., J. Appl. Polymer Sci., 35:755-774 (1988).

4. Formulations for Enteral Administration

Bioactive agents that are not peptides or polypeptides can also beformulated for oral delivery. Oral solid dosage forms are known to thoseskilled in the art. Solid dosage forms include tablets, capsules, pills,troches or lozenges, cachets, pellets, powders, or granules orincorporation of the material into particulate preparations of polymericcompounds such as polylactic acid, polyglycolic acid, etc. or intoliposomes. Such compositions may influence the physical state,stability, rate of in vivo release, and rate of in vivo clearance of thepresent proteins and derivatives. See, e.g., Remington's PharmaceuticalSciences, 21st Ed. (2005, Lippincott, Williams & Wilins, Baltimore, Md.21201) pages 889-964. The compositions may be prepared in liquid form,or may be in dried powder (e.g., lyophilized) form. Liposomal orpolymeric encapsulation may be used to formulate the compositions. Seealso Marshall, K. In: Modern Pharmaceutics Edited by G. S. Banker and C.T. Rhodes Chapter 10, 1979. In general, the formulation will include theactive agent and inert ingredients which protect peptide in the stomachenvironment, and release of the biologically active material in theintestine.

Another embodiment provides liquid dosage forms for oral administration,including pharmaceutically acceptable emulsions, solutions, suspensions,and syrups, which may contain other components including inert diluents;adjuvants such as wetting agents, emulsifying and suspending agents; andsweetening, flavoring, and perfuming agents.

Controlled release oral formulations may be desirable. Non-polypeptidebioactive agents can be incorporated into an inert matrix which permitsrelease by either diffusion or leaching mechanisms, e.g., films or gums.Slowly disintigrating matrices may also be incorporated into theformulation. Another form of a controlled release is one in which thedrug is enclosed in a semipermeable membrane which allows water to enterand push drug out through a single small opening due to osmotic effects.For oral formulations, the location of release may be the stomach, thesmall intestine (the duodenum, the jejunem, or the ileum), or the largeintestine. Preferably, the release will avoid the deleterious effects ofthe stomach environment, either by protection of the active agent (orderivative) or by release of the active agent beyond the stomachenvironment, such as in the intestine. To ensure full gastric resistancean enteric coating (i.e, impermeable to at least pH 5.0) is essential.Examples of the more common inert ingredients that are used as entericcoatings are cellulose acetate trimellitate (CAT),hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55,polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, celluloseacetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. Thesecoatings may be used as mixed films or as capsules such as thoseavailable from Banner Pharmacaps.

III. Uses for Biodegradable, Synthetic Vascular Grafts with IncreasedPatency

The biodegradable, synthetic vascular grafts disclosed herein may beused as venous, arterial or artero-venous conduits for any vascular orcardiovascular surgical application. Exemplary applications include, butare not limited to, congenital heart surgery, coronary artery bypasssurgery, peripheral vascular surgery and angioaccess.

Vascular bypass grafting is most commonly performed for the treatment ofvessel stenosis. However, vascular grafts are also used for thetreatment of other conditions such as arterial aneurysm or chronic renalfailure (as access for hemodialysis). Vascular grafting can be performedby conventional surgery or endovascular techniques.

Coronary artery bypass grafting (CABG) is one example of vascular bypasssurgery. With this procedure, a bypass graft is used to bypass thecoronary artery distal to the site of stenosis or occlusion. When a veingraft is used, one end is anastomosed to the aorta and the other end isanastomosed to the coronary artery beyond the stenosis or occlusion.When an arterial graft is used, the proximal end is left undisturbed(thus preserving the artery's normal blood inflow) and the distal end isanastomosed to the coronary artery beyond the stenosis or occlusion.

Typically, an anastomosis (i.e., the surgical union of tubular parts)between the in situ artery or vein and the synthetic graft is created bysewing the graft to the in situ vessel with suture. Commonly used suturematerials include proline (extruded polypropyline) and ePTFE.

EXAMPLES Example 1. Seeded Human Bone Marrow Mononuclear Cells (hBMCs)Improve the Functional Outcome and Vascular Development of TEVG

Materials and Methods:

Biodegradable PGA-P(CL/LA) Scaffold Construction

A dual cylinder chamber molding system was constructed from a 6.5 mmdiameter polypropylene rod. A 1.4 mm diameter inner cylinder was coredthrough the center of the rod for a length of 30 mm and graduallytapered out to 6.0 mm at the inlet. Polyglycolic acid (PGA) nonwovenfelt (ConcordiaFibers, Coventry, R.I.) was used for the framework of thescaffold. Felts were 300 mm thick with approximately 90% (PGA) or 83%(PLLA) total porosity. The gradual taper of the inner cylinder enabledflat felt sections (6.0 mm×4.0 mm) to be easily shaped into tubes duringtheir insertion through the inlet of the dual cylinder chamber system.Stainless steel needles (21 g) were then introduced into the opposingend to maintain the inner lumen and further compress the felt. A 50:50copolymer sealant solution of 3-caprolactone and L-lactide (P(CL/LA))(263,800 Da, Absorbable Polymers International, Birmingham, Ala.) wascreated by dissolving the copolymer at 5% (w/v) in 1,4-dioxane. TheP(CL/LA) sealant was injected into the inlet of the chamber system andallowed to penetrate the felt and fuse the open seam. The hybridpolyester scaffolds were snap-frozen at −20° C. for 30 minutes to enablerapid transformation of the P(CL/LA) sealant from liquid to solid phase,creating a sealed tube with a newly defined porous structure of P(CL/LA)polymer interconnecting the PGA fibers. The scaffolds were lyophilizedfor 24 hours to eliminate the solvents before removing them from thedual cylinder chamber system.

BMC Isolation

Murine bone marrow was isolated from the femur bones of MCP-1−/− orsyngeneic C57BL/6 mice (Jackson Laboratories). Unfractionated human bonemarrow (10 donors) was purchased from Lonza. Bone marrow was diluted 1:1in sterile PBS and filtered through a 100 μm nylon mesh. Mononuclearcells were then isolated by density gradient centrifugation usinghistopaque-1077 (human) or histopaque-1068 (mouse) (Sigma).

BMC Seeding onto PGA-P(CL/LA) Scaffolds

A fibrin gel solution was used to attach BMC to the scaffold. BMC weresuspended at 2×10⁶ cells/ml in sterile fibrinogen solution (100 mg/mlhuman fibrinogen [Sigma] in PBS). Fifty microliters of BMC-fibrinogensolution (1×10⁶ BMC) was statically seeded onto each scaffold. The cellsolution was solidified onto the scaffold by adding sterile thrombinsolution (100 U/ml human thrombin [Sigma] in 40 mM CaCl₂ in PBS). Seededscaffolds were incubated at 37° C. in RPMI-1640 medium with 10% FBSuntil ready to be surgically implanted. All scaffolds were implantedwithin 48 hours of seeding.

Infrarenal IVC Interposition Surgery

All scaffolds were sutured into the infrarenal inferior vena cava (IVC)of 3-4 month old, female C.B-17 SCID/bg mice (Taconic Farms).Anesthetized mice were placed in the supine position and opened with anabdominal midline incision. Infrarenal IVC or aorta was exposed under 5×magnification, cross-clamped, and excised. Three millimeter lengthscaffolds were then inserted as interposition grafts using a running10-0 nylon suture for the end-to-end proximal and distal anastomoses.Animals were recovered from surgery and maintained without the use ofany anticoagulation or antiplatelet therapy. A total of 97 animals wereimplanted with BMC-seeded, unneeded, or MCP-1 eluting scaffolds. At 1 d,3 d, 1 wk, 3 wk, 6 wk, 10 wk, and 24 wk, animals were sacrificed andgrafts underwent in-vivo perfusion fixation under conditions ofphysiological pressure prior to explantation. All animal experimentswere done in accordance with the institutional guidelines for the useand care of animals, and the institutional review board approved theexperimental procedures described.

Micro-Computed Tomography Angiography (microCTA)

In vivo patency and morphology of the TEVG were evaluated usingmicroCTA. Anesthetized mice were anticoagulated with 100 IU heparinprior to sacrifice. A PE-10 (polyethylene) catheter was inserted intoeither the IVC or aorta and 300 ml of Omnipaque (300 mg/ml) was injectedinto the respective venous or arterial circulatory system. Mice werethen imaged in an X-O™ microCT (Gamma-Medica, Northridge, Calif.).Three-dimensional reconstruction was done using COBRA software (ExximComputing Corporation). Images were combined using Total Commander(Ghisler & Co.) and volume rendered with AMIDE imaging software(Loening).

Histology

Explanted grafts were fixed in 10% formalin overnight and then embeddedin paraffin. Sections were stained with H&E, Gamori trichrome(collagen), and Verhoeff-van Gieson (elastin). Using previouslypublished methods; some pre-implant hBMC-seeded scaffolds were embeddedin glycolmethacrylate to maintain better histologic structure withprocessing.

Morphometric Analysis

Internal diameter and wall thickness were measured for each explantedscaffold using ImageJ software (NIH).

Statistical Analysis

All data are presented as mean±SEM. Statistical significance wascalculated in MS Excel and SPSS 11.5 for Windows. In all multi-groupanalysis overall comparisons between groups were performed with theone-way ANOVA. If a significant difference was found, pair-wisecomparisons were carried out using the Tukey's test, correcting formultiple pair-wise analysis. For strictly pair-wise comparisons,independent student's t-test was carried out. Probability values lessthan 0.05 were regarded as significant.

Results:

Seeding hBMC onto scaffolds significantly improved their patency ratesas IVC interposition grafts compared to unseeded scaffolds implanted inparallel. By post-operative week 10, three out of seven unseededscaffolds were fully occluded, one was >70% stenosed, and the remainingthree were patent (FIG. 1). Micro-CT angiography showed that micecompensated for graft obstruction by developing large venouscollaterals, suggesting that occlusion was likely due to a gradualstenotic process rather than an acute thrombotic event. Confirmatoryhistological analysis of occluded scaffolds showed obstructive cellularin-growth into the lumen. Conversely, when hBMC seeding was performedprior to implantation, 100% patency rates were obtained (FIG. 1). AllhBMC-seeded scaffolds (n=5) were patent at 10 weeks with well-definedinternal lumens as determined by both micro-CT angiography andhistological analysis.

To ensure that hBMC seeding did not merely delay lumen occlusion, threehBMC-seeded scaffolds were followed for 24 weeks to evaluate long-termvascular development and function. All three seeded TEVG remainedpatent, and morphologically and histologically resembled native mouseIVC. The graft wall thickness substantially decreased, leaving an innerlumen and vessel wall comparable to that of native IVC. The originalscaffold material degraded, and had been effectively replaced by amature neovessel with an organized vascular architecture. A clearlydefined intima, consisting of a confluent endothelial monolayer, andmedia, containing 1-2 layers of smooth muscles cells, were present.Circumferentially oriented collagen fibrils made up the supportiveadventitial layer.

Example 2. Stem Cells Constitute a Minor Fraction of Seeded hBMCs

Materials and Methods:

Materials and methods were as described in Example 1 except as describedbelow.

Characterization of hBMCs

Flow cytometry was used to identify and quantify sub-populations in themononuclear cell fraction of human bone marrow. Human BMC from fivedifferent donors were used and tested in triplicate. Antibodies usedwere purchased from BD Biosciences CD8 PerCP(SK1), CD14 FITC (M5E2),CD31 FITC (WM59), CD34 PE and PerCP-Cy5.5 (8G12), CD45 APC-Cy7 (2D1),CD56 PE (NCAM16.2), CD146 (P1H12), CD73 PE (AD2), CD90 FITC (5E10),7AAD; E-Bioscience CD3 APC (HIT3a), CD4 FITC (L3T4), CD19 (PE-Cy7);Miltenyi Biotec CD133 APC (AC133); R&D Systems VEGFR2PE (89106); and AbDSerotec CD105 Alexa 647 (SN6). Cells were acquired on a FACSAria cellsorter and results analyzed using DIVA software (BD Bioscience).

Quantitative Image Analysis

Relative cellularity was measured for each explanted scaffold. Twoseparate sections of each explant were stained with H&E and imaged at400× magnification, noted as a high power field (hpf). The number ofnuclei was then counted in five regions of each section and averaged.Host mouse monocytes and seeded human BM-MNG subpopulations wereidentified by positive F4/80, hCD45, hCD34 and hCD31 expressionrespectively, were also quantified in pre-implant scaffolds and inpost-operative week 1 explants, using similar methods.

Results:

To determine the mechanism by which seeded hBMC improved vascular growthand development of TEVG, the phenotypes and relative abundances of cellpopulations within the human BMC population used for seeding were firstexamined. Human BMC consisted mainly of mature leukocyte populations,including monocytes (1014.7%), CD4⁺ T cells (7.0±2.7%), CD8 T cells(7.9±2.5%), B cells (6.4±2.1%), and NK cells (3.2±1.4) (Table 1).

TABLE 1 Summary of FACS analysis of human BM-MNC Cell Type StainingPanel Percent BMC Late-outgrowth EPC (+) AC133, VEGF-R2 0.0029 ± 0.0042(−) CD45 Early-outgrowth EPC (+) CD14, VEGF-R2 1.7 ± 1.2 Matureendothelial cell (+) CD31, CD146 0.0050 ± 0.024  (−) CD45 Mesenchymalstem cell (+) CD90, CD73,  0.0013 ± 0.00096 CD105 (−) CD45, CD34Hematopoietic stem cell (+) CD34  1.8 ± 0.53 Monocyte (+) CD45, CD14  10± 4.7 CD4 T cell (+) CD3, CD4 7.0 ± 2.7 CD8 T cell (+) CD3, CD8 7.9 ±2.5 B cell (+) CD19 6.4 ± 2.1 Natural killer cell (+) CD56 3.2 ± 1.4Other (neutrophils, 56 ± 10 erythroblasts, etc.)

The largest population of adult stems cells identified was CD34⁺hematopoietic stem cells (HSC; 1.8±0.53%). AC133⁺KDR⁺CD45⁻ cells, likelyto be late-outgrowth endothelial progenitor cells (L-EPC), made up0.0029±0.0042% of the BMC population, while CD90⁺CD73⁺CD105⁺CD45⁻ CD34⁻mesenchymal stem cells (MSC) constituted the smallest percentage of BMC,with yields of only 0.0013±0.00096%. The relative paucity of progenitorcells, smooth muscle cells, and endothelial cells present in the BMCfirst raised the possibility that the seeded BMC may not be the sourceof the cellular constituents of vascular neotissue.

Static seeding, consisting of direct pipetting of a concentratedsuspension of hBMC into scaffolds resulted in a similar distribution ofcell types as in the original population, i.e., there was no obviouspreference for any particular sub-population. Prior to implantation, themajority of hBMC in the scaffold were CD45⁺ mature leukocytes, withgreater than 30 cells per high power field (hpf). CD31⁺ mature EC andCD34⁺ stem cells were also identified, but in much lower numbers, withfewer than 1 cell per hpf respectively. No alpha-smooth muscle actin(αSMA) expression was detectable prior to implantation.

Example 3. Seeded hBMCs are not Incorporated into the DevelopingNeovessel

Materials and Methods:

Materials and methods were as described in Examples 1 and 2 except asdescribed below.

Immunohistochemistry

Species-specific antibodies were used to distinguish between seededhuman cells and recruited murine host cells. Human-specific primaryantibodies used included mouse-anti-human CD31 (Dako), CD68 (Dako), CD34(Abeam), and CD45 (AbD Serotec). Mouse-specific primary antibodies usedincluded rat-anti-mouse Mac-3 (BD Bioscience), F4/80 (AbD Serotec),IL-1beta (R&D Systems), and goat-anti-mouse VEGF-R2 (R&D Systems,minimal cross-reactivity). Primary antibodies that cross-reacted withboth species were mouse-anti-human α-smooth muscle actin (αSMA) (Dako),VEGF (Santa Cruz), and rabbit-anti-human von Willebrand Factor (vWF)(Dako). Antibody binding was detected using appropriate biotinylatedsecondary antibodies, followed by binding of streptavidin-HRP and colordevelopment with 3,3-diaminobenzidine (Vector). Nuclei were thencounterstained with hematoxylin. For immunofluorescence detection, agoat-anti-rabbit IgG-Alexa Fluor 568 (Invitrogen) or a goat-anti-mouseIgG-Alexa Fluor 488 (Invitrogen) was used with subsequent4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstaining.

Detection of Human RNA in TEVG

TEVG harvested 1, 3, 7, or 21 days after implantation or immediatelyprior to implantation were snap-frozen in liquid nitrogen, and RNA wasextracted by mechanical crushing over dry ice followed by incubation inRLT lysis buffer (Oiagen). Samples were passed through Qiashreddercolumns (Oiagen) and processed using RNeasy mini kits (Oigen) accordingto the manufacturer's protocol. Reverse transcription with randomhexamer and oligo-dT primers was performed according to the MultiscribeRT system protocol (Applied Biosystems). PCR reactions were preparedwith TaqMan 2×PCR Master Mix and pre-developed assay reagents fromApplied Biosystems (human GAPDH, Hs99999905 ml. mouse HPRT, Mm00446968ml) and analyzed on an iQ5 (Bio-Rad). Species-specificity of the humanand mouse probes was confirmed on human and mouse control arterysegments. To determine the limits of detection of human RNA, standardcurves were generated by measuring human GAPDH levels obtained from10-fold serial dilutions of human BMC cells in culture and human BMCcells seeded onto scaffolds. The limit of detection of this assay wasbetween 10 and 100 cells.

Results:

To determine if seeded hBMC or their progeny were directly contributingto the cellularity of the developing TEVG, these cells were next trackedover 24 weeks using human-specific markers and immunohistochemistry.CD34⁺ human stem cells could no longer be detected by post-operativeweek 1, but CD68⁺ human monocytes and CD31⁺ human EC could still bedetected within the scaffold wall. Neither cell type, however, wasidentified along the luminal surface, the location of vascular neotissueformation. By 3 weeks, no human cells expressing CD45, CD68, CD31, orCD34 could be detected anywhere in the grafts, suggesting that seededhBMC were not likely present after this time point. Human specific GAPDHRNA detection via Q-RT-PCR confirmed the presence of human RNA on theTEVG prior to implantation and failed to detect any human RNA after the7 POD (FIG. 2).

Although seeded hBMC were not permanently incorporated into thedeveloping neovessel, they did affect the cellular development of thescaffold. Total cellularity of both hBMC-seeded and unseeded scaffoldsprogressively increased over 10 weeks, with the most substantial changeoccurring during the first week of development. At post-operative days 1and 3, no significant differences in cellularity could be detectedbetween hBMC-seeded scaffolds and unseeded scaffolds. However, by day 7,the increase in cellularity was significantly greater for hBMC-seededscaffolds (n=6; 270±22 cells/hpf) compared to unseeded scaffolds (n=6;160±40 cells/hpf) (p<0.001). This increased cellularity was primarilydue to a significantly increased infiltration of host mouse monocytesinto hBMC-seeded scaffolds (120±20 monocytes/hpf), as compared tounseeded scaffolds (60±12 monocytes/hpf) (p<0.001) (FIG. 3).

Example 4. BMC-Derived MCP-1 is a Critical Molecule in TEVG Development

Materials and Methods:

Materials and methods were as described in Examples 1-3 except asdescribed below.

ELISA for Cytokine and Chemokine Profile

hBMC were cultured at 2×10⁶ cells/ml on either PGA-P(CL/LA) scaffold ortissue culture plastic for 48 hrs in RPMI-1640+10% FBS. VEGF, SDF-1,IL-1β, and MCP-1 levels in the supernatants were measured using ELISAkits from R&D Systems. Minimum detection values were SDF-1 alpha 156pg/mL, VEGF 15.6 pg/mL, IL-1beta 3.9 pg/mL and MCP-1 31.2 pg/mL.

Results:

Based on the above findings, it was hypothesized that host monocytespromoted neovessel development. The remaining examples address howseeded hBMC could increase early host monocyte recruitment. It was firstexamined how interactions with scaffold biomaterials affected productionof chemotactic factors by hBMC. ELISA was used to test for the presenceof several candidate chemokines including: interleukin-1beta (IL-1β),MCP-1, stromal derived factor-1alpha (SDF-1α) and vascular endothelialgrowth factor (VEGF). Exposure to scaffold was found to significantlyincrease the amount of interleukin-1beta (IL-1β) and MCP-1 secreted byhBMC (FIGS. 4A and 4B). The production of stromal derived factor-1alpha(SDF-1α) and vascular endothelial growth factor (VEGF), two factorsfrequently associated with stem cell recruitment, either with or withoutexposure to scaffold material was below the level of detection of theELISA.

Knowing that MCP-1 is a potent stimulant for monocyte recruitment, thenext series of experiments was performed to determine if the MCP-1produced by the seeded BMC was important in TEVG development. To analyzethe effects of BMC-derived MCP-1, scaffolds were seeded with murine BMC(mBMC) obtained from either MCP-1−/− mice or syngeneic C57BL/6 wild type(WT) mice. BMC from MCP-1−/− mice are unable to produce MCP-1, whichspecifically eliminates the role of MCP-1 secreted from seeded BMC.After 10 wks of development as IVC interposition grafts in the SCID/bgmouse, MCP-1−/− mBMC-seeded scaffolds (n=5) were compared to WTmBMC-seeded scaffolds (n=5). Internal lumen diameters were decreased,with a trend towards significance (0.55±0.12 mm vs. 0.71±0.12 mm;p=0.07) and graft wall thicknesses were significantly increased(0.25±0.04 mm vs. 0.20±0.01 mm; p=0.04) in scaffolds seeded withMCP-1−/− BMC, suggesting that seeded BMC-derived MCP-1 was playing acritical role in TEVG development (FIGS. 5A and 5B).

Example 5. MCP-1 Microparticles Mimic Paracrine Function of Seeded hBMCs

Materials and Methods:

Materials and methods were as described in Examples 1-4 except asdescribed below.

Synthesis of MCP-1 Eluting Materials

Recombinant human MCP-1 (R&D Systems) was encapsulated intobiodegradable alginate microparticles using previously published methods(Jay, et al., FASEB Jour., (2008)). Microparticles were incorporatedinto the scaffold by directly mixing them into the P(CL/LA) sealant at aconcentration of 50 ug/ul. Scaffolds were then constructed using similarmethods to those described above. The size and shape distribution of theMCP-1 microparticles, on and off the scaffold, were imaged using a XL-30scanning electron microscope (FEI Company). The release profile of MCP-1from these scaffolds was measured by ELISA (R&D Systems). MCP-1 elutingscaffolds (n=5) were each immersed in 1 ml of M199 medium and incubatedin a 37° C. orbital shaker. At 1, 2, 4, 8, 12, 24, 48, 72, 96, 120, 144,and 168 hours, medium was collected and replaced with 1 ml of fresh M199medium. Media samples were stored at −20° C. until analysis.

Results:

To investigate whether MCP-1, administered alone, could effectivelyinduce early monocyte recruitment and improve outcomes for unseededscaffolds, a system was created that enabled delivery of MCP-1 directlyfrom the scaffold without seeding hBMC. Recombinant human MCP-1 wasencapsulated into biodegradable alginate microparticles, 1-20 μm indiameter, making them comparable in size to the heterogeneous populationof hBMC. The microparticles were then embedded into the scaffold tomimic the MCP-1 releasing function of seeded hBMC. Embeddedmicroparticles released approximately 200 ng of MCP-1 from the scaffoldover the course of 72 hours, which was similar to the duration ofretention of the majority of seeded hBMC (FIG. 6).

Unseeded scaffolds embedded with MCP-1 microparticles developed andfunctioned similarly to hBMC-seeded scaffolds when implanted as IVCinterposition grafts in SCID/bg mice. Host monocyte recruitment at 1week was significantly increased in MCP-1 eluting scaffolds (n=3; 201±62monocytes/hpf), compared to both hBMC-seeded (n=6; 120±17 monocytes/hpf;p<0.01) and unseeded (n=4; 60±12 monocytes/hpf; p<0.01) scaffolds (FIG.7A). Furthermore, after 10 weeks of development, all MCP-1 elutingscaffolds (n=5) were patent with internal lumen diameters comparable tohBMC-seeded scaffolds (n=7) (0.53±0.08 mm vs. 0.63±0.19 mm; p=0.30)(FIG. 7B). Both the seeded scaffolds and those with MCP-1 elutingabilities had better patency rates than the unseeded scaffolds. WhileMCP-1 microparticles did maintain the internal luminal structure, thewall thickness of MCP-1 eluting scaffolds (0.31±0.02 mm) was morecomparable to unseeded scaffolds (0.35±0.10 mm; p=0.48) than hBMC-seededscaffold (0.20±0.03 mm; p<0.001) (FIG. 7C).

Example 6. G-CSF Administration Promotes Early Outward Remodeling inTEVG

Materials and Methods:

Materials and methods were as described in Examples 1-5 except asdescribed below.

G-CSF Administration

Unseeded scaffolds were implanted as IVC interposition grafts andserially monitored with ultrasound over an 8-week time course. Theexperimental group received preoperative administration of G-CSF (10μg/kg) while the control group did not.

Ultrasound Interrogation

Ultrasonography was utilized to serially examine functional andmorphologic changes of tissue engineering graft. Mice were anesthetizedwith 1.5% isoflurane. The internal diameter and wall thickness will beserially measured at longitudinal section of the graft using Vevo 770(Visualsonics) over the described time course. Measurements wereobtained at three locations including measurements at the proximal,middle and distal thirds of the graft in addition to a specificmeasurement at the narrowest and widest portion of the graft. Allmeasurements were performed by a single operator with expertise in mousevascular ultrasonography and repeated in triplicate to minimize operatordependent variation.

Statistical Analysis

All data are presented as mean±SEM. Statistical significance wascalculated in MS Excel and SPSS 11.5 for Windows. In all multi-groupanalysis overall comparisons between groups were performed with theone-way ANOVA. If a significant difference was found, pair-wisecomparisons were carried out using the Tukey's test, correcting formultiple pair-wise analysis. For strictly pair-wise comparisons,independent student's t-test was carried out. Probability values lessthan 0.05 were regarded as significant.

Results:

Preoperative administration of G-CSF promotes outward remodeling. Serialultrasound monitoring demonstrated significantly wider lumens at alltime points between 2 and 8 weeks after implantation. These findingswere most marked at 4 weeks when the G-CSF group internal diametermeasured 0.84 mm±0.06 mm and the control group measured 0.68 mm±0.05 mm(p<0.05) (FIGS. 8A and 8B).

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

We claim:
 1. A method for increasing the patency of a vascular graftcomprising a biodegradable polymer, the method comprising incorporatinginto or onto the graft monocyte chemoattractant protein 1 (MCP-1),wherein the MCP-1 is locally released from the graft in an effectiveamount following graft implantation to recruit an effective amount ofhost monocytes to the graft within one week of graft implantation toprevent, inhibit or reduce stenosis, and promote neotissue formation andincrease the patency of the graft in the host over time relative to thepatency of the graft in the absence of MCP-1.
 2. The method of claim 1,wherein the biodegradable or bioabsorbable polymers are selected fromthe group consisting of poly(lactic acid), poly(glycolic acid),polyanhydrides, poly(ortho)esters, polyesters, polyurethanes, poly(buticacid), poly(valeric acid), poly(caprolactone), poly(hydroxyalkanoates),and poly(lactide-co-caprolactone), or combinations, blends orco-polymers thereof.
 3. The method of claim 1, wherein the biodegradableor bioabsorbable polymers are formed into a fiber-based mesh.
 4. Themethod of claim 3, wherein the fiber-based mesh is a non-woven mesh. 5.The method of claim 4, wherein the vascular graft further comprises apolymeric sealant.
 6. The method of claim 5, wherein the polymericsealant comprises a co-polymer of ε-caprolactone and L-lactide.
 7. Themethod of claim 1, further comprising administering to the host, orincorporating into graft an additional cytokine or chemokine selectedfrom the group consisting of interleukin (IL)1α, IL-1β, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12(p40), IL-12(p70), IL-13,IL-15, IL-17, IP-10, eotaxin, interferon γ (IFNγ), granulocytecolony-stimulating factor (G-CSF), granulocyte/macrophagecolony-stimulating factor (GM-CSF), macrophage inflammatory protein 1α(MIP-1α), RANTES, tumor necrosis factor (TNF)-α, platelet-derived growthfactor (PDGF)-AA, PDGF-AB/BB, TGF-beta, VEGF, and combinations thereof.8. The method of claim 7, wherein the cytokine or chemokine is IL-1β orG-CSF.
 9. The method of claim 7 further comprising administering G-CSFto the host.
 10. The method of claim 9 wherein the administration ofG-CSF occurs prior to implantation of the graft in the host.
 11. Themethod of claim 1 wherein the effective amount of MCP-1 is released frommicroparticles in the graft over a period of 1 to 3 days after the graftis implanted into the host.
 12. The method of claim 1 wherein the MCP-1is incorporated into or onto the graft by seeding the graft withmicroparticles comprising MCP-1 before implantation of the graft intothe host.
 13. The method of claim 1 further comprising seeding the graftwith human bone marrow mononuclear cells prior to implantation of thegraft.
 14. The method of claim 1 wherein the MCP-1 is provided inmicroparticles between 1 μm and 20 μm in diameter incorporated into thegraft.
 15. The method of claim 1 wherein the internal diameter of thegraft is larger relative to the internal diameter of the graft in theabsence of MCP-1 ten weeks after implantation of the graft into thehost.
 16. The method of claim 1 wherein the wall thickness of the graftis thinner relative to the wall thickness of the graft in the absence ofMCP-1 ten weeks after implantation of the graft into the host.
 17. Themethod of claim 1 wherein the monocytes enhance neotissue formation andreduce stenosis at the site of graft implantation as the graft degrades.18. The method of claim 1, wherein the host is a pediatric patient.